Publisher Correction: Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

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Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

A contribution of epigenetic modifications to B cell tolerance has been proposed but not directly tested. Here we report that deficiency of ten-eleven translocation (Tet) DNA demethylase family members Tet2 and Tet3 in B cells led to hyperactivation of B and T cells, autoantibody production and lupus-like disease in mice. Mechanistically, in the absence of Tet2 and Tet3, downregulation of CD86, which normally occurs following chronic exposure of self-reactive B cells to self-antigen, did not take place. The importance of dysregulated CD86 expression in Tet2- and Tet3-deficient B cells was further demonstrated by the restriction, albeit not complete, on aberrant T and B cell activation following anti-CD86 blockade. Tet2- and Tet3-deficient B cells had decreased accumulation of histone deacetylase 1 (HDAC1) and HDAC2 at the Cd86 locus. Thus, our findings suggest that Tet2- and Tet3-mediated chromatin modification participates in repression of CD86 on chronically stimulated self-reactive B cells, which contributes, at least in part, to preventing autoimmunity.

Loop-extruding Smc5/6 organizes transcription-induced positive DNA supercoils.

The structural maintenance of chromosomes (SMC) protein complexes-cohesin, condensin, and the Smc5/6 complex (Smc5/6)-are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6's recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled DNA at cohesin-dependent loop boundaries on budding yeast (Saccharomyces cerevisiae) chromosomes. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes and efficiently initiate loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.

Highly rigid H3.1/H3.2-H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells.

The placenta is a highly evolved, specialized organ in mammals. It differs from other organs in that it functions only for fetal maintenance during gestation. Therefore, there must be intrinsic mechanisms that guarantee its unique functions. To address this question, we comprehensively analyzed epigenomic features of mouse trophoblast stem cells (TSCs). Our genome-wide, high-throughput analyses revealed that the TSC genome contains large-scale (>1-Mb) rigid heterochromatin architectures with a high degree of histone H3.1/3.2-H3K9me3 accumulation, which we termed TSC-defined highly heterochromatinized domains (THDs). Importantly, depletion of THDs by knockdown of CAF1, an H3.1/3.2 chaperone, resulted in down-regulation of TSC markers, such as Cdx2 and Elf5, and up-regulation of the pluripotent marker Oct3/4, indicating that THDs maintain the trophoblastic nature of TSCs. Furthermore, our nuclear transfer technique revealed that THDs are highly resistant to genomic reprogramming. However, when H3K9me3 was removed, the TSC genome was fully reprogrammed, giving rise to the first TSC cloned offspring. Interestingly, THD-like domains are also present in mouse and human placental cells in vivo, but not in other cell types. Thus, THDs are genomic architectures uniquely developed in placental lineage cells, which serve to protect them from fate reprogramming to stably maintain placental function.

Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.

ATF7-Dependent Epigenetic Changes Are Required for the Intergenerational Effect of a Paternal Low-Protein Diet.

Paternal dietary conditions may contribute to metabolic disorders in offspring. We have analyzed the role of the stress-dependent epigenetic regulator cyclic AMP-dependent transcription factor 7 (ATF7) in paternal low-protein diet (pLPD)-induced gene expression changes in mouse liver. Atf7+/- mutations cause an offspring phenotype similar to that caused by pLPD, and the effect of pLPD almost vanished when paternal Atf7+/- mice were used. ATF7 binds to the promoter regions of ∼2,300 genes, including cholesterol biosynthesis-related and tRNA genes in testicular germ cells (TGCs). LPD induces ATF7 phosphorylation by p38 via reactive oxygen species (ROS) in TGCs. This leads to the release of ATF7 and a decrease in histone H3K9 dimethylation (H3K9me2) on its target genes. These epigenetic changes are maintained and induce expression of some tRNA fragments in spermatozoa. These results indicate that LPD-induced and ATF7-dependent epigenetic changes in TGCs play an important role in paternal diet-induced metabolic reprograming in offspring.

The transcription factor ATF7 mediates lipopolysaccharide-induced epigenetic changes in macrophages involved in innate immunological memory.

Immunological memory is thought to be mediated exclusively by lymphocytes. However, enhanced innate immune responses caused by a previous infection increase protection against reinfection, which suggests the presence of innate immunological memory. Here we identified an important role for the stress-response transcription factor ATF7 in innate immunological memory. ATF7 suppressed a group of genes encoding factors involved in innate immunity in macrophages by recruiting the histone H3K9 dimethyltransferase G9a. Treatment with lipopolysaccharide, which mimics bacterial infection, induced phosphorylation of ATF7 via the kinase p38, which led to the release of ATF7 from chromatin and a decrease in repressive histone H3K9me2 marks. A partially disrupted chromatin structure and increased basal expression of target genes were maintained for long periods, which enhanced resistance to pathogens. ATF7 might therefore be important in controlling memory in cells of the innate immune system.

The maintenance of chromosome structure: positioning and functioning of SMC complexes.

Structural maintenance of chromosomes (SMC) complexes, which in eukaryotic cells include cohesin, condensin and the Smc5/6 complex, are central regulators of chromosome dynamics and control sister chromatid cohesion, chromosome condensation, DNA replication, DNA repair and transcription. Even though the molecular mechanisms that lead to this large range of functions are still unclear, it has been established that the complexes execute their functions through their association with chromosomal DNA. A large set of data also indicates that SMC complexes work as intermolecular and intramolecular linkers of DNA. When combining these insights with results from ongoing analyses of their chromosomal binding, and how this interaction influences the structure and dynamics of chromosomes, a picture of how SMC complexes carry out their many functions starts to emerge.

Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination.

Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.

The replication checkpoint protects fork stability by releasing transcribed genes from nuclear pores.

Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.

Histone H3.3 chaperone HIRA renders stress-responsive genes poised for prospective lethal stresses in acquired tolerance.

Appropriate responses to environmental challenges are imperative for the survival of all living organisms. Exposure to low-dose stresses is recognized to yield increased cellular fitness, a phenomenon termed hormesis. However, our molecular understanding of how cells respond to low-dose stress remains profoundly limited. Here we report that histone variant H3.3-specific chaperone, HIRA, is required for acquired tolerance, where low-dose heat stress exposure confers resistance to subsequent lethal heat stress. We found that human HIRA activates stress-responsive genes, including HSP70, by depositing histone H3.3 following low-dose stresses. These genes are also marked with histone H3 Lys-4 trimethylation and H3 Lys-9 acetylation, both active chromatin markers. Moreover, depletion of HIRA greatly diminished acquired tolerance, both in normal diploid fibroblasts and in HeLa cells. Collectively, our study revealed that HIRA is required for eliciting adaptive stress responses under environmental fluctuations and is a master regulator of stress tolerance.

Sororin mediates sister chromatid cohesion by antagonizing Wapl.

Sister chromatid cohesion is essential for chromosome segregation and is mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin is thought to mediate cohesion. In vertebrates, Sororin is essential for cohesion and stable cohesin-DNA interactions, but how Sororin performs these functions is unknown. We show that DNA replication and cohesin acetylation promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's ability to dissociate cohesin from DNA. Sororin has only been identified in vertebrates, but we show that many invertebrate species contain Sororin-related proteins, and that one of these, Dalmatian, is essential for cohesion in Drosophila. The mechanism we describe here may therefore be widely conserved among different species.

Cohesin Ubiquitylation and Mobilization Facilitate Stalled Replication Fork Dynamics.

Replication fork integrity is challenged in conditions of stress and protected by the Mec1/ATR checkpoint to preserve genome stability. Still poorly understood in fork protection is the role played by the structural maintenance of chromosomes (SMC) cohesin complex. We uncovered a role for the Rsp5Bul2 ubiquitin ligase in promoting survival to replication stress by preserving stalled fork integrity. Rsp5Bul2 physically interacts with cohesin and the Mec1 kinase, thus promoting checkpoint-dependent cohesin ubiquitylation and cohesin-mediated fork protection. Ubiquitylation mediated by Rsp5Bul2 promotes cohesin mobilization from chromatin neighboring stalled forks, likely by stimulating the Cdc48/p97 ubiquitin-selective segregase, and its timely association to nascent chromatids. This Rsp5Bul2 fork protection mechanism requires the Wpl1 cohesin mobilizer as well as the function of the Eco1 acetyltransferase securing sister chromatid entrapment. Our data indicate that ubiquitylation facilitates cohesin dynamic interfacing with replication forks within a mechanism preserving stalled-fork functional architecture.

Biallelic variants in GTF3C5, a regulator of RNA polymerase III-mediated transcription, cause a multisystem developmental disorder.

General transcription factor IIIC subunit 5 (GTF3C5) encodes transcription factor IIIC63 (TFIIIC63). It binds to DNA to recruit another transcription factor, TFIIIB, and RNA polymerase III (Pol III) to mediate the transcription of small noncoding RNAs, such as tRNAs. Here, we report four individuals from three families presenting with a multisystem developmental disorder phenotype with biallelic variants in GTF3C5. The overlapping features include growth retardation, developmental delay, intellectual disability, dental anomalies, cerebellar malformations, delayed bone age, skeletal anomalies, and facial dysmorphism. Using lymphoblastoid cell lines (LCLs) from two affected individuals, we observed a reduction in TFIIIC63 protein levels compared to control LCLs. Genome binding of TFIIIC63 protein is also reduced in LCL from one of the affected individuals. Additionally, approximately 40% of Pol III binding regions exhibited reduction in the level of Pol III occupancy in the mutant genome relative to the control, while approximately 54% of target regions showed comparable levels of Pol III occupancy between the two, indicating partial impairment of Pol III occupancy in the mutant genome. Yeasts with subject-specific variants showed temperature sensitivity and impaired growth, supporting the notion that the identified variants have deleterious effects. gtf3c5 mutant zebrafish showed developmental defects, including a smaller body, head, and eyes. Taken together, our data show that GTF3C5 plays an important role in embryonic development, and that biallelic variants in this gene cause a multisystem developmental disorder. Our study adds GTF3C5-related disorder to the growing list of genetic disorders associated with Pol III transcription machinery.

Transient Methionine Deprivation Triggers Histone Modification and Potentiates Differentiation of Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) require high levels of methionine (Met). Met deprivation results in a rapid decrease in intracellular S-adenosyl-methionine (SAM), poising human iPSCs for differentiation and leading to the apoptosis of undifferentiated cells. Met deprivation triggers rapid metabolic changes, including SAM, followed by reversible epigenetic modifications. Here, we show that short-term Met deprivation impairs the pluripotency network through epigenetic modification in a 3D suspension culture. The trimethylation of lysine 4 on histone H3 (H3K4me3) was drastically affected compared with other histone modifications. Short-term Met deprivation specifically affects the transcription start site (TSS) region of genes, such as those involved in the transforming growth factor ß pathway and cholesterol biosynthetic process, besides key pluripotent genes such as NANOG and POU5F1. The expression levels of these genes decreased, correlating with the loss of H3K4me3 marks. Upon differentiation, Met deprivation triggers the upregulation of various lineage-specific genes, including key definitive endoderm genes, such as GATA6. Upon differentiation, loss of H3K27me3 occurs in many endodermal genes, switching from a bivalent to a monovalent (H3K4me3) state. In conclusion, Met metabolism maintains the pluripotent network with histone marks, and their loss potentiates differentiation.

Genome-organizing factors Top2 and Hmo1 prevent chromosome fragility at sites of S phase transcription.

Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gammaH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1-dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.

Attaching Accessory Devices to the Replisome.

Evidence mounts, via two studies published in Molecular Cell (Villa et al. 2016; Samora et al. 2016), that Ctf4 recruits to the replisome various factors that play diverse roles in chromosome duplication, by acting as an interaction hub.

Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer.

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non-cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

Inhibition of HDAC8 Reduces the Proliferation of Adult Neural Stem Cells in the Subventricular Zone.

In the adult mammalian brain, neurons are produced from neural stem cells (NSCs) residing in two niches-the subventricular zone (SVZ), which forms the lining of the lateral ventricles, and the subgranular zone in the hippocampus. Epigenetic mechanisms contribute to maintaining distinct cell fates by suppressing gene expression that is required for deciding alternate cell fates. Several histone deacetylase (HDAC) inhibitors can affect adult neurogenesis in vivo. However, data regarding the role of specific HDACs in cell fate decisions remain limited. Herein, we demonstrate that HDAC8 participates in the regulation of the proliferation and differentiation of NSCs/neural progenitor cells (NPCs) in the adult mouse SVZ. Specific knockout of Hdac8 in NSCs/NPCs inhibited proliferation and neural differentiation. Treatment with the selective HDAC8 inhibitor PCI-34051 reduced the neurosphere size in cultures from the SVZ of adult mice. Further transcriptional datasets revealed that HDAC8 inhibition in adult SVZ cells disturbs biological processes, transcription factor networks, and key regulatory pathways. HDAC8 inhibition in adult SVZ neurospheres upregulated the cytokine-mediated signaling and downregulated the cell cycle pathway. In conclusion, HDAC8 participates in the regulation of in vivo proliferation and differentiation of NSCs/NPCs in the adult SVZ, which provides insights into the underlying molecular mechanisms.

Codependency and mutual exclusivity for gene community detection from sparse single-cell transcriptome data.

Single-cell RNA-seq (scRNA-seq) can be used to characterize cellular heterogeneity in thousands of cells. The reconstruction of a gene network based on coexpression patterns is a fundamental task in scRNA-seq analyses, and the mutual exclusivity of gene expression can be critical for understanding such heterogeneity. Here, we propose an approach for detecting communities from a genetic network constructed on the basis of coexpression properties. The community-based comparison of multiple coexpression networks enables the identification of functionally related gene clusters that cannot be fully captured through differential gene expression-based analysis. We also developed a novel metric referred to as the exclusively expressed index (EEI) that identifies mutually exclusive gene pairs from sparse scRNA-seq data. EEI quantifies and ranks the exclusive expression levels of all gene pairs from binary expression patterns while maintaining robustness against a low sequencing depth. We applied our methods to glioblastoma scRNA-seq data and found that gene communities were partially conserved after serum stimulation despite a considerable number of differentially expressed genes. We also demonstrate that the identification of mutually exclusive gene sets with EEI can improve the sensitivity of capturing cellular heterogeneity. Our methods complement existing approaches and provide new biological insights, even for a large, sparse dataset, in the single-cell analysis field.